There is compelling evidence for variability in the severity of Cystic Fibrosis (CCF) disease amongst CF patients and differences in the nature of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene mutation cannot account for all of this variation. Using a murine model of CF, our group has shown that secondary genes can ameliorate the severe intestinal phenotype,, the major cause of mortality in this species. These modifier genes have not yet been identified. However, as our comparative studies of the intestinal mucosa of CF mice with mild disease and CF mice with severe disease show a correlation between disease amelioration and chloride ion secretion, we suggest that disease modification may occur through modulation of the chloride channel(s) on the apical membrane of intestinal epithelial cells is a fundamental to developing our understanding of how disease severity can be ameliorated in intestinal epithelial cells. So far, our findings suggest a correlation between ClC-2 expression and intestinal chloride secretion. Specifically, we found that chloride (Cl) secretion by intestinal mucosa obtained from CF mice with mild disease can be blocked by inhibitors of ClC-2 function and the ClC-2 protein is localized toward the apical pole of these epithelial cells. Hence, ClC-2 protein is appropriately localized to mediate chloride secretion. However, ClC-2 can only mediate secretion if it is activated in epithelial cells and so far, we have only a poor understanding of how ClC-2 channel activity is regulated. Our overall goals of this study are to understand how ClC-2 is regulated and to determine if this channel protein can mediate secretion in intestinal epithelial tissue. Specific Goals: 1) to study the regulation of ClC-2 channel function in vitro and in vivo. We have determined that purified, reconstituted ClC-2 protein can mediate Cl ion electrodiffusion. Our reconstitution system will permit detailed analysis of its permeation and gating properties. We also found that Clc-2 is endogenously localized at the apical surface of the Caco-2 cell line ane have developed tools to study its regulation in the model epithelial cell line. 2) to determine whether ClC-2 in Cl secretion in Caco-2 cells by modifying its expression through transfection. Furthermore, the role of ClC- 2 in secretion by native tissues will be investigated by generating transgenic mice over-expressing ClC-2.